铜绿假单胞菌耐药性在生物被膜形成过程中的变化

Change of intrinsic drug resistance in the formation of Pseudomonas aeruginosa biofilms

目的 研究铜绿假单胞菌遗传性耐药(gyrA基因突变和MexAB OprM主动外排)在生物被膜形成过程中对药物抗性的作用。方法 通过PCR技术鉴定gyrA基因突变株和MexAB OprM主动外排株,利用三亲杂交的方法将绿色荧光蛋白(GFP)基因转到铜绿假单胞菌药物敏感株中,测定gyrA基因突变株、MexAB OprM主动外排株以及带有绿色荧光蛋白的铜绿假单胞菌在特氟隆上产生生物被膜耐药的规律。结果 筛选出gyrA基因突变株和MexAB OprM主动外排株。通过三亲杂交成功构建了带有pGFPuv质粒的铜绿假单胞菌。将突变株和敏感株培养在最低杀菌浓度(MBC)的环丙沙星溶液中形成生物被膜使菌株的存活率均在50%以上。结论 比较遗传性耐药和生物被膜耐药的作用表明gyrA基因突变和主动外排在生物被膜耐药形成前期发挥主要作用,当生物被膜完全形成之后,铜绿假单胞菌的耐药性有可能是由生物被膜介导的。

Objective To screen gyrA mutant and multidrug resistance (MDR) efflux pump (MexAB-OprM) of Pseudomonas aeruginosa (Pa) and to identify the contribution of gyrA mutant and MDR efflux pump to bacterial drug resistance during the formation of Pa biofilms. Methods The strains of gyrA mutant and MDR efflux pump were clinical isolates and detected by PCR. Tri-relative hybridization experiments were performed to transfer report gene of GFP into sensitive Pa strains. The formation of bacterial biofilms was observed by scanning electron microscope followed by analysis of bacterial survival rate. Results The fluorescent strains with pGFPuv could form mature biofilms on Teflon surface. Over a period of 72 h, the survival rate of biofilm bacteria was more than 50% in its′ MBC of ciprofloxacin. Conclusion The express of gyrA mutation and MDR efflux played major roles in Pa resistance during the initial stage of biofilm development. But after 72 h of incubation, there was no significant difference between mutants and sensitive strains in terms of survival rate.